Cas9-mediated gene targeting

The Cas9-mediated generation of transgenic mice can be achieved by the injection of Cas9 protein or mRNA together with a guide RNA (gRNA) that determines the specificity, and a targeting vector into a mouse zygote. The enzyme will introduce DNA breaks at the location predetermined by the co-injected gRNA. As long as a targeting vector is co-injected these breaks can also be repaired by homologous recombination (HR) leading to defined gene modifications. This is typically used to introduce loxP sites or larger cassettes such as GFP or Cre. 

This service offers the generation of knock in mice by pronuclear injection of components of the CRISPR/Cas9 system and recombination substrates. Injections are routinely performed in C57BL6/J background, with other backgrounds possible on request (price may vary). The embryo transfers following microinjections are performed under SPF conditions, thus allowing founders to be introduced into most facilities. Please contact us directly for special requests such as injections in other mouse strains.

1. A completely filled out Request Form
2. The GMO datasheet as requested by the veterinary office
3. An iRATS breeding project opened under researchers own license to be used for weaned offspring (iRATS users only)
4. The genebank (*.gbk or *.gb) file with the sequence of interest marking the KI region and a genebank file of the modified locus.
5. 10 μg of a PCR product spanning the KO region at > 150 ng/μl
6. 50 μg of the targeting vector (circular) at a concentration of 1µg/μl
7. A validated genotyping PCR approach (for detecting 5' insertion and 3' insertion as well as the gene of interest), tested with WT genomic DNA spiked with the linearized transgene at various concentrations

The cost of this service is 11500 CHF for inbred (C57BL/6J) mouse strains. This fee covers the donor animals, the injection of CRISPR/Cas9 components including the targeting vector, embryo transfers, collection of biopsies, hygiene test and subsequent animal care costs until the founders are turned over to the researcher. An additional fee of 700 CHF will be charged when founders should already be bred to wt animals, before transfer to the TCF.

For terms and conditions please click  here.

In case of questions, please contact Johannes vom Berg before submitting a microinjection request. Every Friday we offer a “Targeting Club” at 9:00 am in our seminar rooms (Wagistr. 12, Schlieren or Sternwartstrasse 6, Unispital, Zürich), where you are welcome to discuss your project with us (see also upcoming dates).